All cells are covered with a dense and complex array of sugar chains. Sialic acids (Sias) are a family of nine-carbon sugars that are typically present at the outermost units of these chains. By virtue of their terminal position, sialic acids act as binding sites for many exogenous and endogenous receptors such as the Influenza viruses and the Siglic family of endogenous proteins. Such sugars are thus useful drug targets for the prevention and treatment of infection. They are also involved in various biological and pathological processes such as neuronal plasticity and cancer metastasis. In many of these instances, the precise structures of the sialic acid and the residues it is attached to play critical roles. Thus, studying sialic acid functions is of great biological importance. In addition, sialic acids can be taken up from certain dietary sources (red meat and dairy products), and may also be associated with certain disease states, such as cancer and heart disease.
Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) converts the sialic acid N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc.) In non-human mammals, Neu5Gc is recognized by a number of endogenous binding proteins, as well as by pathogenic organisms such as bacteria and viruses. Humans are unable to produce endogenous Neu5Gc because of an evolutionary inactivating mutation in their CMAH gene. Specifically, this mutation involves a frame-shifting exon deletion of 92 base pairs in the 5′ region that gives rise to a truncated protein that lacks the amino acid residues that are necessary for enzymatic activity (Schlenzka, W., et al., FEBS Lett. (1996) 385: 197-200.) This mutation occurred sometime after the divergence of humans from their last common ancestor, so humans are the only known animals missing a functional CMAH gene (Chou, H-H, et al., Proc. Nat. Acad. Sci. (2002), 99 (18): 11736-11741.) Although the cause for this mutation is unknown, it may have been caused by negative selection of individuals that were CMAH+, because of the recognition of Neu5Gc by pathogens.
Neu5Gc is known to be immunogenic in humans (Noguchi A., et al., J. Biochem. Tokyo (1995), 117 (1): 59-62.) Such immunogenicity is believed to play a role in the immune response observed in humans that come into contact with mammalian products, such as cosmetics, food, mammalian cells and cell products, as well as therapeutic agents derived from non-human mammals or exposed to non-human mammalian products. Attempts have been undertaken to try to diminish the Neu5Gc content of recombinantly produced human glycoproteins in cell lines by altering the cell lines using RNAi to suppress expression of the CMAH gene (Chenu S., et al., Biochim. Biophys. Acta. (2003), 1622 (2): 133-144.)
However, there remains a need to produce biological products for human use that lack Neu5Gc, such as the production of human cells or tissues in the absence of Neu5Gc medium, and by using transgenic non-human mammals lacking a fully functional CMAH gene to produce Neu5Gc products for human use.